PROJECT SUMMARY/ABSTRACT Patients with head and neck squamous cell cancer (HNSCC) continue to have poor prognosis, largely because of disease recurrence and/or the development of metastatic disease. New therapies are an unmet need for patients with recurrent/metastatic HNSCC. Immune-based therapies, including immune checkpoint inhibitors (ICIs), have been introduced and Pembrolizumab, an anti-PD-1 monoclonal antibody, is currently approved by the US Food and Drug Administration (FDA) for the first-line treatment of patients with unresectable recurrent/metastatic HNSCC. Use of ICIs is based on the premise that rejuvenation of suppressed anti-tumor immunity in cancer patients will improve outcome. Unfortunately, only a minority of the HNSCC patients treated with ICIs responds to the immunotherapy (IT), possibly due to the unresponsiveness of immune cells to activation in profoundly immunosuppressed HNSCC patients. Exosomes have emerged as major contributors to tumor- associated immune suppression and a significant barrier to antibody-based and adoptive cell-based therapies in cancer. We have reported that IT is not effective in cancer patients with high levels of circulating exosomes, and have also shown that circulating exosomes carrying immunosuppressive ligands, such as PD-L1, impair functions of immune effector cells in HNSCC patients and negatively impact disease progression. Therefore, we hypothesize that the removal of immunosuppressive exosomes from the circulation of HNSCC patients prior to IT will promote immune cell recovery and significantly increase the response rate. To test this hypothesis and develop novel therapeutic capabilities for cancer patients, we have established a partnership between Aethlon Medical, Inc. and three academic institutions. Aethlon Medical, Inc. has developed the Hemopurifier, a good manufacturing practices (GMP)-compatible device for removal of blood-borne viruses and exosomes and designed for use with standard dialysis machines. In Aim 1, we will characterize exosomes removed from HNSCC patients? plasma by the research-grade version of the Hemopurifier. We will correlate the immunosuppressive profiles, functions and miRNA content of these exosomes with clinicopathological endpoints and disease activity. In addition, using a mouse model, we will demonstrate that depletion of exosomes restores anti-tumor immunity and inhibits tumor growth, while delivery of suppressive exosomes promotes tumor growth and carcinogenesis. In Aim 2, we will conduct a single-arm Phase II clinical trial using a clinical-grade Hemopurifier to establish that removal of exosomes from the circulation of patients with recurrent/metastatic HNSCC improves responses to IT. Finally, in Aim 3, we will utilize data and samples from the Phase II clinical trial to identify predictors of benefit from exosome depletion by Hemopurifier prior to IT. Successful completion of the proposed project is expected to result in novel therapeutic capabilities and new predictive tools based on plasma exosomes to improve clinical responses to ITs for patients with HNSCC.